PROJECT SUMMARY/ABSTRACT Diagnosing mosquito-borne diseases, such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV), in the early stage of infection is crucial in limiting the spread among the population (through bites of infected mosquitos and sexual transmission) as well as preventing serious illness to the patient. Many early- stage diagnostic devices have been developed in recent years to detect pathogens with improved sensitivity and simpler/cheaper devices, but they fail to improve the medical logistical system for early diagnosis of infectious disease, due to the labor-intensive sample preparation process. Simple, low-cost, robust sample collection and sample processing tools requiring no power or skilled labor are currently in demand to enable early diagnosis of infectious disease. We propose to design and fabricate a new blood-sampling syringe that fully incorporates a nucleic acid purification function. This innovative idea uses the vacuum created by the plunger in the syringe to continuously perform blood lysis, silica absorption, washing, and stabilization tasks. If successful, a simple draw of blood from patients will result in a completely prepared sample that is ready for nucleic acid detection. Also, a small, handheld, and robust real-time polymerase chain reaction (PCR) device, capable of multiplexed detection of ZIKV, DENV, and CHIKV, will be developed to work directly with the integrated syringe. The real-time PCR device will be designed to work without external power for several hours and is simple to operate by inserting the integrated syringe and a cartridge. The multiplexed detection of ZIKV, DENV, and CHIKV will be enabled by the automated excitation/emission filter turret, and the device can analyze nucleic acid amplifications to determine the quantity of pathogenic nucleic acid in the sample to reveal the stage of infection. The portable, handheld real-time PCR will be fabricated with low-cost materials to allow mass production at low cost and enable a large distribution of the device. The estimated material cost for each real-time PCR device is ~300 USD. This project will produce a comprehensive two-step diagnosis platform, consisting of an integrated syringe, real- time PCR device, and cartridges. The platform is operated by simply drawing blood from a patient using the integrated syringe and inserting the syringe into the real-time PCR device, which results in a transferring of purified viral RNAs from the syringe into the cartridge. The cartridge holds lyophilized real-time PCR reagents to enable nucleic acid amplification as soon as the RNA is eluted into the cartridge. The two-step diagnosis platform is unique in its capability of comprehensively replicating the standard infectious disease diagnosis process, currently used in clinics and hospitals, and is enabled by the automation of sample preparation in the syringe. The proposed project could potentially make a transformative impact to global health in handling infectious disease diagnosis by enabling early-stage diagnostics.